This invention is concerned generally with protein purification and specifically with the purification of fibrinogen from the milk of transgenic animals using cation exchange chromatography.
Fibrinogen, the main structural protein in the blood responsible for the formation of clots exists as a dimer of three polypeptide chains; the Axcex1 (66.5 kD), Bxcex2 (52 kD) and xcex3 (46.5 kD) are linked through 29 disulphide bonds. The addition of asparagine-linked carbohydrates to the Bxcex2 and xcex3 chains results in a molecule with a molecular weight of 340 kD. Fibrinogen has a trinodal structure, a central nodule, termed the E domain, contains the amino-termini of all 6 chains including the fibrinopeptides (Fp) while the two distal nodules termed D domains contain the Carboxy-termini of the Axcex1, Bxcex2 and xcex3 chains. Fibrinogen is proteolytically cleaved at the amino terminus of the Axcex1 and Bxcex2 chains releasing fibrinopeptides A and B (FpA and FpB) and converted to fibrin monomer by thrombin, a serine protease that is converted from its inactive form by Factor Xa. The resultant fibrin monomers non-covalently assemble into protofibrils by DE contacts on neighboring fibrin molecules. This imposes a half staggered overlap mode of building the fibrin polymer chain. Contacts are also established lengthwise between adjacent D domains (DD contacts) leading to lateral aggregation. Another serine protease, Factor XIII is proteolytically cleaved by thrombin in the presence of Ca2+ into an activated form. This activated Factor XIII (Factor XIIIa) catalyses crosslinking of the polymerised fibrin by creating isopeptide bonds between lysine and glutamine side chains. The first glutamyl-lysyl bonds to form are on the C-terminal of the xcex3 chains producing Dxe2x80x94D crosslinks. Subsequently, multiple crosslinks form between adjacent Axcex1 chains, the process of crosslinking imparts on the clot both biological stability (resistance to fibrinolysis) and mechanical stability [Sienbenlist and Mosesson, Progressive Cross-Linking of Fibrin y chains Increases Resistance to Fibrinolysis, Journal of Biological Chemistry, 269: 28414-2841, 1994].
The coagulation process can readily be engineered into a self sustained adhesive system by having the fibrinogen and Factor XIII as one component and thrombin and Ca2+ as the second component which catalysis the polymerization process. These adhesion systems, know in the art as xe2x80x9cFibrin Sealentsxe2x80x9d or xe2x80x9cFibrin Tissue Adhesivesxe2x80x9d have found numerous application in surgical procedures and as delivery devices for a range of pharmaceutically active compounds [Sierra, Fibrin Sealent Adhesive Systems: A Review of Their Chemistry, Material Properties and Clinical Applications, Journal of Biomaterials Applications, 7:309-352, 1993; Martinowitz and Spotnitz, Fibrin Tissue Adhesives, Thrombosis and Haemostasis, 78:661-666, 1997; Radosevich et al., Fibrin Sealent:Scientific Rationale, Production Methods, Properties and Current Clinical Use, Vox Sanguinis, 72:133-143, 1997].
It has been estimated that the annual US clinical need for fibrin sealents is greatly in excess of the 300 kg/year that can be harvested using the current cryoprecipitation methods used by plasma fractionaters. Alternative sources of fibrinogen, by far the major component in fibrin sealant, have therefore been explored with recombinant sources being favored [Butler et al., Current Progress in the Production of Recombinant Human Fibrinogen in the Milk of Transgenic animals, Thrombosis and Haemostasis, 78: 537-542, 1997]. While cell culture systems have demonstrated the ability to produce small amounts (1-4 ug/ml) of fibrinogen, it has been shown that mammals are capable of producing transgenic human fibrinogen at levels of up to 5.0 g/L in their milk making this a commercially viable method for the production of human fibrinogen [Prunkard et al., High-level expression of recombinant human fibrinogen in the milk of transgenic mice, Nature Biotechnology, 14:867-871, 1996; Cottingham et al., Human fibrinogen from the milk of transgenic sheep. In: Tissue Sealants: Current Practice, Future Uses. Cambridge Institute, Newton Upper Falls, Mass., Mar. 30-Apr. 2, 1996 (abstract)].
Differences have been identified between recombinant human fibrinogen and fibrinogen which has been purified from human plasma. Fibrinogen which has been purified from human plasma has two alternately spliced gamma chains (xcex3 and xcex3xe2x80x2). In contrast, recombinant human fibrinogen only has the major form xcex3. Further, the glycosylation of the beta and gamma chains (there is no N-linked glycosylation of the alpha chain) of recombinant human fibrinogen differs slightly from that on plasma derived fibrinogen, but is similar to the glycosylation found on other proteins expressed in the milk of transgenic animals. In addition, the Ser3 of the alpha chain of recombinant human fibrinogen is more highly phosphorylated than Ser3 of the alpha chain of plasma derived fibrinogen, although the difference in phosphorylation does not result in functional differences. Also, there are detectable differences in heterogeneity caused by C-terminal proteolysis of a number of highly protease-sensitive sites on the alpha chain. Differences of a similar magnitude are also observed between plasma-derived fibrinogen from different sources.
Another driving force for the development of totally recombinant fibrin-based sealents stems from the fact that commercially available adhesives originate from pooled plasma. As blood-derived products have been associated with the transmission of human immunodefieciency virus (HIV), hepatitis virus and other etiological agents, the acceptance and availability of such adhesives is limited. While the incorporation of viral removal and inactivation procedures has increased the safety of these products (for example, U.S. Pat. Nos. 4,960,757 and 5,116,950), plasma derived fibrinogen is still not without risk. The use of autologous plasma reduces the risk of disease transmission; however, autologous adhesives can only be used in elective surgery when the patient is able to donate the blood in advance.
While the main use of fibrinogen is thought to be for the preparation of adhesive or sealing agents, fibrinogen also has other applications in the field of medicine, for example as a coating for polymeric articles as disclosed in U.S. Pat. No. 5,272,074, and the concern for safety apply to any of these other uses in medicine.
Various methods for the purification of fibrinogen have been described where, in most cases, the starting material has been either plasma or more usually cryoprecipitate or Cohn Fraction 1, both of which are rich in fibrinogen. Schwartz et al., [U.S. Pat. Nos. 4,362,567; 4,377,572; 4,414,976] have disclosed a process for the manufacture of fibrinogen tissue adhesive using cryoprecipitation of plasma as the major purification step. Burnouf et al., (1990) [Biochemical and Physical Properties of a Solvent-Detergent-Treated Fibrin Glue, Vox Sang 58:77-84] describe a process for the preparation of fibrinogen concentrate from 150 L of human plasma. After thawing anticoagulated plasma at 37xc2x0 C., the plasma was subjected to a 10% ethanol precipitation. Following dissolution of the precipitated fibrinogen a Solvent-Detergent viral inactivation technique was used followed by a further two ethanol precipitation procedures to remove the Solvent-Detergent chemicals. The final material is quoted as being 93.5% pure with respect to protein content and is sold under the trade name Biocoll(copyright).
Vila et al., (1985) [A Rapid Method for Isolation of Fibrinogen From Human Plasma by Precipitation with PEG 6000, Thrombosis Research 39:651-656] describe a method whereby fibrinogen is precipitated from citrated plasma using an 8% solution of PEG 6000. Following dissolution the fibrinogen is then further precipitated using 2 mol/l acetic acid-acetate buffer pH 4.6. A final precipitation with ammonium sulphate is used to give a product with 93xc2x16% clottable protein and in 60xc2x19% yield. Using similar techniques, Masri et al., (1983) [Isolation of Human Fibrinogen of High Purity and in High Yield using PEG 1000, Thrombosis and Haemostasis, 49:116-119] have also purified fibrinogen from thrombin-free plasma using precipitation with PEG 1000. A three stage precipitation technique was used leading to a product with 98% clottable protein in 65-70% yield.
It is evident from the literature that the purification of fibrinogen from plasma is usually accomplished using precipitation techniques. In many examples this leads to relatively pure product but it is doubtful that a process based solely on precipitation could be used in the purification of human fibrinogen from milk. Fibrinogen has a propensity for binding other plasma proteins including but not exclusively plasminogen, tPA, factor XIII and fibronectin which are often co-purified during precipitation techniques. Numerical data of some of these proteins are often quoted in manufacturers specifications. This contamination can be tolerated in human plasma-derived fibrinogen, which is destined to be used in humans, as the contaminating human proteins would probably not be antigenic in humans. However, it is unacceptable for high levels of these proteins to be present in a human fibrinogen product manufactured from non-human animal milk, as these proteins, from the milk of a different species, would probably be expected to be antigenic in humans. Hence it is anticipated that any purification scheme from milk would include several chromatographic techniques.
The purification of fibrinogen from human plasma using column chromatography has been described by Dempfle and Keen (1987) [Purification of Human Plasma Fibrinogen by Chromatography On Protamine Agarose, Thrombosis and Haemostasis 46:19-27]. Fibrinogen was recovered in a single-step operation in 65-80% yield with  greater than 90% clottability. The use of Protamine agarose is well known in the art and has been used extensively at a small scale for purification of fibrinogen from plasma and from recombinant sources including mammalian cell culture [Lord et al., Purification and characterization of recombinant human fibrinogen, Blood Coagulation and Fibrinolysis 4:55-59, 1993] and yeast [Roy et al., Secretion of Biologically Active Recombinant Fibrinogen by Yeast, Journal of Biological Chemistry, 270:23761-23767, 1995). However, even though the technique is very specific, the expense of the chromatography media and the suspected lability of the biological ligand render it nonviable on a commercial large-scale. Other examples of very specific chromatographic steps are available in the literature including the use of peptide ligands [Kuyas et al., Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on GlyProArgProLys-Fractogel, Thrombosis and Haemostasis, 63:439-444, 1990] and even the use of columns containing monoclonal antibodies [Takebe et al., Calcium Ion Dependent Monoclonal Antibody Against Human Fibrinogen: Preparation, Characterization and Application to Fibrinogen Purification, Thrombosis and Haemostasis, 73:662-667, 1995]. However as discussed above, the use of such expensive and delicate chromatographic materials remains cost-prohibitive at an industrially enabling scale.
As an alternative to the use of biological ligands, anion exchange chromatography has been practiced during the purification of fibrinogen from plasma, although this has been done mostly at an analytical scale where its incorporation is to subfractionate fibrinogen species [Kuyas et al., A Subfraction of Human Fibrinogen With High Sialic Acid Content and Elongated xcex3 Chains, Journal of Biological Chemistry, 257:1107-1109, 1982]. An anion exchange purification technique for fibrinogen has been disclosed by Bernard et al. (EP 0 555 135 A1), in which fibrinogen is bound from treated cryoprecipitate in 120 mM NaCl, 10 mM Tris, pH 8.8 and eluted at 200 mM NaCl, 10 mM Tris, pH 8.8. This procedure leads to greater selectivity in terms of the eluted fibrinogen.
In developing a purification process from transgenic milk it is generally accepted that eliminating casein is a high priority (Wilkins and Velander, xe2x80x9cIsolation of Recombinant Proteins from Milkxe2x80x9d, Journal of Cellular Biochemistry 49:333-338 [1992]). Casein is a mixture of mostly insoluble proteins present as micellar suspensions (the remainder of proteins in milk are called whey proteins). Casein micelles are not filterable and readily block process equipment. This property prevents milk from being processed by conventional chromatography columns before casein removal or solubilisation. This has inspired the recent development of techniques for casein removal or solubilisation. For example Denman [xe2x80x9cIsolation of Components of Interest From Milkxe2x80x9d, PCT WO 94/19935] has disclosed a method for solubilising casein micelles by the addition of positively charged agents. This results in making the milk more amenable for processing. However, this technique does not remove casein, and the agents used are prohibitively expensive for large scale processing. Kutzko et al. [xe2x80x9cPurification of Biologically Active Peptides From Milkxe2x80x9d, PCT WO 97/42835] have disclosed a method for the separation of a soluble milk component from milk using tangential flow filtration. This technique involves adding a chelating agent to the milk to solubilise casein before applying to ultrafiltration apparatus whereby the component of interest is collected in the permeate and the solubilised casein remains in the retentate. This technique has also been useful with milk in which the casein fraction has not been solubilised. However, it is expected that this technique is more suited to peptides and small proteins. Furthermore, this technique although useful in providing a casein-free solution, does not offer significant purification of the transgenic protein unless it is linked with another unit operation, for example, chromatography. Therefore it is apparent that while milk can be more amenable to processing using the techniques described above, a process comprising simultaneous casein removal and fibrinogen purification would have significant economic advantages.
On a small scale, elimination of casein can be achieved by centrifugation; however, as the centrifugation force needs to be very high this is not a viable technique for industrial application. Historically, casein removal in the food industry has been carried out either by the addition of rennet or by the use of acid; both of which result in casein precipitation [Swaisgood, Developments in Dairy Chemistryxe2x80x941: Chemistry of Milk Protein, Applied Science Publishers, N.Y., 1982]. Again these are not generally applicable or preferred techniques for the purification of human proteins from milk e.g. renin is animal derived and would have to be obtained from a controlled source that is pathogen free. Precipitation by acid, while being cost-effective and efficient, is not generally applicable, as the low pH may result in degradation or precipitation of the human protein unless the human protein is acid stable.
Precipitation of caseins with low concentrations of salts and polymeric precipitating agents has been described in the literature. For example approximately 17% sodium sulphate and 22% ammonium sulphate have been used to precipitate casein micelles before further fractionation of whey proteins [Swaisgood, Developments in Dairy Chemistryxe2x80x941: Chemistry of Milk Protein, Applied Science Publishers, NY, 1982]. Lee and Antonsen [PCT WO 97/09350] have disclosed that precipitation of casein with PEG is used as an early step in the purification of transgenic alpha-1-antitrypsin from sheep milk. Similarly, Wilkins and Velander, (1992) [Isolation of Recombinant Proteins from Milk, Journal of Cellular Biochemistry 49:333-338] advocate the use of PEG precipitation of casein as an early step in transgenic protein purification.
With respect to the purification of fibrinogen from milk, a major obstacle is the separation of the casein fraction. Fibrinogen and casein micelles share several characteristics, most notably their relatively insoluble nature and propensity for self-aggregation. They can also both be regarded as relatively hydrophobic with a tendency to bind non-specifically to both other proteins and surfaces. The similar properties shared between casein and fibrinogen is also illustrated by the fact that both are readily purified by precipitation. From the evidence in the literature therefore, it would be unlikely that precipitation could be solely employed as a stand alone technique for the removal of fibrinogen from casein. Consequently, a method for fibrinogen purification from milk which combined both casein removal and fibrinogen purification would be a significant advantage.
Thus there remains a need in the art for a method of obtaining purified fibrinogen from milk (which transgenic origin gives the fibrinogen the advantages of significant yields and safety in terms of viral non-contamination), the method in its preferred embodiment providing highly purified, non-antigenic fibrinogen in a simple, cost-effective and efficient manner. A major obstacle is the separation of fibrinogen from casein, which is difficult due to certain similarities in their properties.
Cation ion exchange chromatography (CEX) is a separation technique which exploits the interaction between positively charged groups on a protein and negatively charged groups on a substrate. As pH influences charges on proteins, the pH of the medium in which CEX is carried out greatly influences the separation performance. CEX substrates can be grouped into 2 major types; those which maintain a negative charge on the substrate over a wide pH range (strong CEX substrates) and those which maintain a negative charge on the substrate over a narrow pH range (weak CEX). Strong cation exchange substrates usually incorporate sulphonic acids derivatives as functional groups (e.g. Sulphonates, S-type or Sulphopropyl groups, SP-types). In order to maximize the performance of the CEX substrate, in terms of binding, the pH of the medium in which the separation is carried out is usually below the isoelectric point of the protein to be bound (the isoelectric point or pI of a protein is that pH value at which the protein carries no net charge, at pH values above the pI, the protein has a net negative charge and at pH values below the pI, the protein has a net positive charge and will be bound to a CEX substrate). CEX has been used quite extensively in the separation of milk proteins particularly the casein fraction. For example, Law et al., [Quantitative Fractionation of Ovine Casein by Cation-Exchange FPLC, Milchwissenchaft, 45: 279-282, 1992] have described the resolution of casein into subfamilies using a Mono S(trademark) column at pH 5.0. Similarly, Leaver and Law [Preparative-scale Purification of Bovine Caseins on a Cation Exchange Resin, Journal of Dairy Research, 59: 557-561, 1992] have described the separation of bovine casein from acid precipitated whole casein on S-Sepharose Fast Flow(trademark) at pH 5.0. Whey proteins have also been separated by CEX chromatography. For example, Hahn et al., [Bovine Whey Fractionation based on Cation-Exchange Chromatography, Journal of Chromatography A, 795: 277-287, 1997] describe the separation of the major whey proteins at pH 4.6 using milk which was acidified to remove the casein fraction. This method, however, is inappropriate for the purification of fibrinogen from milk, as the casein removal by acidification would precipitate fibrinogen.
The isolation of lactoferrin from milk using CEX is disclosed in WO 95/22258 (Gene Pharming Europe BV). However, the teaching of WO 95/22258 would not be considered applicable for the purification of fibrinogen due to the differences in the properties of lactoferrin and fibrinogen, and moreover, the elevated ionic strength conditions under which the milk is contacted with the CEX resin in WO 95/22258 would result in fibrinogen not binding to the CEX resin.
The use of CEX in the purification of fibrinogen has not been considered appropriate as fibrinogen is reported to have a pI of 5.5 and can therefore be described as an acidic protein more suited to anion exchange chromatography [Marguerie et al., The Binding of Calcium to Bovine Fibrinogen, Biochimica et Biophysica Acta, 490: 94-103, 1977; Weisel and Cederholm-Williams, Fibrinogen and Fibrin: Characterization, Processing and Applications, Handbook of Biodegradable Polymers (Series: Drug Targetting and Delivery) 7:347-365, 1997]. The problem of casein removal for an acidic protein was highlighted by Van Cott et al (Affinity Purification of Biologically Active and Inactive Forms of Recombinant Human Protein C Produced in Porcine Mammary Gland, Journal of Molecular Recognition, 9: 4407-414 [1996]) who describe previous significant losses of acidic protein C if conventional centrifugation or precipitation steps are used to remove the casein fraction. Solubilisation of casein micelles using EDTA followed by anion exchange chromatography still resulted in casein contamination of human protein C. Up to 20% contamination of protein C by casein following, even highly selective immunoaffinity chromatography, was evidnent if high salt concentrations were not used prior to the immunoaffinity chromatography step. The consequences of using high salt in this step were that up to 20% of the protein C did not bind to the immunoaffinity column.
Of some surprise therefore to the inventors was the fact that fibrinogen can be bound to CEX substrates at pH values above the reported pI of the protein directly from milk and that this attribute can be used to develop a very effective purification technique. The majority of milk proteins, including casein, being relatively acidic in nature, can be prevented from binding to the CEX column or can be resolved from the fibrinogen by the use of changes in pH or ionic strength. Such a finding constitutes a part of this invention. A further advantage of this technique is that prior removal of the casein fraction, by the use of precipitation techniques favored by practitioners, is not necessary. This advantage is thought to provide a significant economic incentive to the incorporation of CEX in a purification protocol for the manufacture of fibrinogen from transgenic non-human sources.
This patent application describes techniques whereby Cation Exchange Chromatography is used in the purification of transgenic fibrinogen from milk. The techniques can be applied to whole milk, skimmed milk or a milk fraction using a variety of chromatography contacting modes. As the prior removal of casein is not required, the use of precipitation agents and centrifigation equipment can be avoided and therefore process economics can be improved. Fibrinogen may be collected at a high yield (up to 95%) in a very pure state (up to 90%) with very little casein contamination and so the step may be considered as incorporating casein removal and substantial fibrinogen purification into a single unit operation. The fibrinogen requires no further purification or only subsequent minor purification using conventional protein purification techniques.
This invention is based on the finding that cation exchange chromatography can be employed, at a pH value higher than the reported pI of fibrinogen, in the purification of fibrinogen from milk. The present application describes, inter alia, a process wherein cation exchange chromatography is employed at a pH value higher than the reported pI of fibrinogen in the purification of fibrinogen from milk, thus allowing for the substantial purification of fibrinogen while simultaneously removing the casein fraction. In the present invention, the need for expensive filtration equipment as outlined by Kutzko (WO 97/42835) is not necessary for the removal of insoluble milk components. Before cation exchange chromatography, the milk is simply delipidated using commercial continuous flow centrifuges which are ubiquitous in the milk processing industry. As such, the cation exchange chromatography purification procedure integrates casein removal and substantial fibrinogen purification into a single unit operation and this is believed to provide a significant economic incentive to adoption of this technique. More specifically, milk, which may contain a chelating agent or other agent capable of disrupting casein micelle structure, is contacted with a CEX substrate under conditions of pH which favour adsorption of fibrinogen but discourage binding of milk proteins. The substrate is then washed and eluted with a series of buffers of suitable pH and/or ionic strength resulting in the resolution of fibrinogen from milk proteins in a substantially purified form. The technique can be applied to whole milk, skimmed milk or a milk fraction.